Project Summary/Abstract Mycobacterium abscessus (Mab) is a rapidly growing non-tuberculous mycobacterium (NTM) that causes a wide range of illnesses including lung, skin and soft-tissue infections, as well as disseminated disease. Treatment of Mab infections is difficult because the bacterium is intrinsically resistant to many classes of antibiotics. Thus, there is a need to develop new therapies against Mab infection. The mycobacterial cell wall is a popular target for antibiotics as its biogenesis is essential for bacterial growth. While cell wall synthesis has been extensively studied in M. tuberculosis (Mtb), relatively little is known about how Mab builds its cell wall and how this process differs from Mtb?s. To identify components of cell wall synthesis with unique roles in Mab physiology, I performed genome-wide transposon mutagenesis coupled with high throughput-sequencing on Mab. I then compared the essentiality of cell wall enzymes between Mab and Mtb. The data reveals Mab3167c, a predicted penicillin-binding-lipoprotein (PBP-lipo), as being essential in Mab, while its homolog in Mtb is non- essential. Mab3167c is predicted to be a transpeptidase that cross-links segments of the foundational peptidoglycan (PG) layer of the cell wall. My preliminary data shows that repressing PBP-lipo impairs bacterial growth and leads to gross morphological abnormalities in the cell. Given that PG synthesis has not been studied in Mab, nor has the function of PBP-lipo, this proposal seeks to answer two central questions: 1) What is the role of PBP-lipo in Mab PG synthesis? and 2) What cell wall enzymes genetically and physically interact with PBP- lipo in Mab? Aim 1 interrogates the localization and function of PBP-lipo during PG synthesis using time-lapse microscopy. With this approach, I will determine in real time where PBP-lipo localizes in the cell and assess how its depletion influences PG synthesis. Aim 2 seeks to identify the functional genetic and physical network of PBP-lipo in Mab. Previous work from our lab demonstrated that Mtb cell wall enzymes have unique sets of genetic interactions and work in protein complexes to coordinate PG synthesis in a spatially and temporarily coordinated manner. Using CRISPR-interference, I will knock down cell wall enzymes in combination with PBP- lipo to determine which pairs genetically interact. I will also perform immunoprecipitation assays to identify putative binding partners of PBP-lipo. All together, this work will elucidate when and where PBP-lipo functions in the cell as well as illuminate how this enzyme contributes to PG synthesis. Moreover, this work will identify cell wall synthesis genes that genetically interact with PBP-lipo as well as uncover proteins that function in complex with the enzyme. These experiments will help uncover the specificities of Mab PG synthesis and cell wall construction. Ultimately, my findings will not only advance the knowledge of cell wall biology in NTMs, but also provide key insights into new drug targets and inform the development of successful treatments for Mab infection. ! !